Method and apparatus for selective biological material detection

ABSTRACT

The present invention relates to bioassay materials useful for the detection of toxic substances and, more particularly, to packaging materials for food and other products, along with methods for their manufacture and use. The invention provides a unique composite material capable of detecting and identifying multiple biological materials within a single package. The biological material identification system is designed for incorporation into existing types of flexible packaging material such as polyvinylchloride or polyolefin films, and its introduction into the existing packaging infrastructure will require little or no change to present systems or procedures.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 09/218,827, filedon Dec. 22, 1998 and now U.S. Pat. No. 6,051,388, having an issue dateof Apr. 18, 2000, the contents of which is herein incorporated byreference.

FIELD OF THE INVENTION

This invention relates to the detection of pathogenic microorganisms, orbiological materials, and more particularly relates to a compositebioassay material useful for the detection of particular toxicsubstances, its method of manufacture and method of use, wherein thecomposite material is particularly useful for food packaging and thelike, and is capable of simultaneously detecting and identifying amultiplicity of such biological materials.

BACKGROUND OF THE INVENTION

Although considerable effort and expense have been put forth in aneffort to control food borne pathogenic microorganisms, therenevertheless exist significant safety problems in the supply of packagedfood. For example, numerous outbreaks of food poisoning brought about byfoodstuffs contaminated with strains of the E-Coli, Campylobacter,Listeria, Cyclospora and Salmonella microorganisms have caused illnessand even death, not to mention a tremendous loss of revenue for foodproducers. These and other microorganisms can inadvertently taint food,even when reasonably careful food handling procedures are followed. Thepossibility of accidental contamination, for example by temperatureabuse, in and of itself, is enough to warrant incorporation of safe andeffective biological material diagnosis and detection procedures.Further complicating the situation is the very real possibility that aterrorist organization might target either the food or water supply of amunicipality or even a nation itself, by attempting to include apathogenic microorganism or toxic contaminant capable of causingwidespread illness or even death. If, by accident or design, the foodsupply of a particular population were to be contaminated, it is notonly imperative that the population be alerted to the contamination, butit is further necessary that the particular contaminant be quickly andprecisely pinpointed so that appropriate countermeasures may be taken.

Thus, if it were possible to readily substitute standard packagingmaterials with a flexible material capable of

1) quickly and easily detecting the presence, and

2) indicating the particular identity of a variety of pathogenicbiological materials, a long felt need would be satisfied.

DESCRIPTION OF THE PRIOR ART

The Berkeley Lab Research News of Dec. 10, 1996 in an article entitled“New Sensor Provides First Instant Test for Toxic E. Coli Organism”reports on the work of Stevens and Cheng to develop sensors capable ofdetecting E. Coli strain 0157:H7. A color change from blue to redinstantaneously signals the presence of the virulent E. Coli 0157:H7microorganism. Prior art required test sampling and a 24 hour cultureperiod in order to determine the presence of the E. Coli microorganism,requiring the use of a variety of diagnostic tools including dyes andmicroscopes. An alternative technique, involving the use of polymerasechain reaction technology, multiplies the amount of DNA present in asample until it reaches a detectable level. This test requires severalhours before results can be obtained. The Berkeley sensor is inexpensiveand may be placed on a variety of materials such as plastic, paper, orglass, e.g. within a bottle cap or container lid. Multiple copies of asingle molecule are fabricated into a thin film which has a two partcomposite structure. The surface binds the biological material while thebackbone underlying the surface is the color-changing signaling system.

The Berkeley researchers do not teach the concept of incorporating anymeans for self-detection within food packaging, nor do they contemplatethe inclusion of multiple means capable of both detecting andidentifying the source of pathogenic contamination to a technicallyuntrained end user, e.g. the food purchaser or consumer.

Wang et al, in an article entitled “An immune-capturing andconcentrating procedure for Escherichia coli 0157:H7 and its detectionby epifluorescence microscopy” published in Food Microbiology, 1998,Vol. 15 discloses the capture of E. coli on a polyvinylchloride sheetcoated with polyclonal anti-E. coli 0157:H7 antibody and stained withfluorescein-labeled anti-E. coli 0157:H7. After being scraped from thePVC surface, the cells were subjected to epifluorescence microscopy fordetermining presence and concentration. The reference fails to teach orsuggest the concept of incorporating any means for self-detection withinfood packaging, nor does it contemplate the inclusion of multiple meanscapable of both detecting and identifying the source of pathogeniccontamination to a technically untrained end user, e.g. the foodpurchaser or consumer, and especially fails to disclose such detectionwithout the use of specialized detection techniques and equipment.

U.S. Pat. No. 5,776,672 discloses a single stranded nucleic acid probehaving a base sequence complementary to the gene to be detected which isimmobilized onto the surface of an optical fiber and then reacted withthe gene sample denatured to a single stranded form. The nucleic acidprobe, hybridized with the gene is detected by electrochemical oroptical detection methodology. In contrast to the instantly disclosedinvention, this reference does not suggest the immobilization of theprobe onto a flexible polyvinylchloride or polyolefin film, nor does itsuggest the utilization of gelcoats having varying porosities to act asa control or limiting agent with respect to the migration of antibodiesor microbial material through the bioassay test material, or to serve asa medium for enhancement of the growth of the microbial material.

U.S. Pat. No. 5,756,291 discloses a method of identifying oligomersequences. The method generates aptamers which are capable of binding toserum factors and all surface molecules. Complexation of the targetmolecules with a mixture of nucleotides occurs under conditions whereina complex is formed with the specific binding sequences but not with theother members of the oligonucleotide mixture. The reference fails tosuggest the immobilization of the aptamers upon a flexiblepolyvinylchloride or polyolefin base material, nor does it suggest theuse of a protective gelcoat layer which acts as a means to selectivelycontrol the migration of antibodies and antigens, or to serve as amedium for enhancement of the growth of microbial material.

SUMMARY OF THE INVENTION

The present invention relates to packaging materials for food and otherproducts, along with methods for their manufacture and use. The presenceof undesirable biological materials in the packaged material is readilyascertained by the consumer, merchant, regulator, etc. under ordinaryconditions and without the use of special equipment. A multiplicity ofbiological materials threaten our food supply. The present inventionprovides a unique composite material capable of detecting andidentifying multiple biological materials within a single package. Thebiological material identification system is designed for incorporationinto existing types of flexible packaging material such aspolyvinylchloride and polyolefin films, and its introduction into theexisting packaging infrastructure will require little or no change topresent systems or procedures. Thus, the widespread inclusion of thebiological material detecting system of the instant invention will beboth efficient and economical.

In one embodiment of the invention the biological material detectingsystem prints a pattern containing several antibodies or aptamers,derived from plant or animal origins, onto a packaging material which isusually a type of polymeric film, preferably a polyvinylchloride orpolyolefin film and most preferably a polyethylene film which hasundergone a surface treatment, e.g. corona discharge to enhance thefilm's ability to immobilize the antibodies upon its surface. The agentsare protected by a special abrasion resistant gel coat in which theporosity is tailored to control the ability of certain antibodies, toxicsubstances, etc. to migrate therethrough. Each antibody is specific to aparticular biological material and is printed having a distinctive iconshape. The detection system may contain any number of antibodies capableof detecting a variety of common toxic food microbes; although anynumber of microbes may be identified via the inventive concept taughtherein, for the purpose of this description, the microbes of interestwill be limited to E. Coli, Salmonella, Listeria and Cyclospora.

An important feature of the biological material detection system is itsall-encompassing presence around and upon the product being packaged.Since the biological material detecting system is designed as anintegral part of 100% of the packaging material and covers all surfacesas utilized, there is no part of the packaged product which can beexposed to undetected microbes. In the past, the use of single locationor in situ detectors have left a majority of the area around and uponthe packaged product exposed to undetected microbes. This greatlyincreased the chance that a spoiled or tainted product might beinadvertently consumed before the toxic agent had spread to the locationof the in situ detector. The biological material detection system of thepresent invention avoids this problem by providing a plurality ofindividual detectors per unit area which are effective to insurepositive detection of any pathogenic microorganisms within the productbeing tested. In order to be effective a particular degree ofsensitivity is required, e.g. the detecting system must be capable ofpositively identifying one microbial cell in a 25 gram meat sample In apreferred embodiment, four detectors per square inch of packagingmaterial surface have been utilized, and in a most preferred embodimentnine or more detectors per square inch are incorporated upon the film'ssurface.

By use of the biological material detection system of the presentinvention a packager or processor can independently determine themultiplicity and identity of those biological materials against whichthe packaged product is to be protected. Although it is envisioned thatthe large majority of biological material detection treated packagingwill be generic to approximately four of the most common microbes, thesystem will nevertheless allow each user to customize the protectionoffered to the public.

The biological material detecting system will not merely detect thepresence of biological materials, it will also identify the particularbiological materials located in a packaged product. This unique featureallows for the immediate identification of each particular biologicalmaterial present since the antibodies are specific to a detector havinga definitive icon shape or other identifying characteristic. Althoughthe end use consumer is primarily interested in whether a food productis, or is not, contaminated per se, the ability to detect and identifythe particular biological material immediately is of immeasurable valueto merchants, processors, regulators and health officials. The abilityto immediately identify a toxic material will lead to greatly reducedresponse times to health threats that might be caused by the biologicalmaterial and will also enhance the ability for authorities to locate thesource of the problem. The biological material detecting system of thepresent invention exhibits an active shelf life in excess of 1 yearunder normal operating conditions. This enhances the use of a biologicalmaterial detection system on products which are intended to be storedfor long periods of time. If these products are stored so as to be readyfor immediate use in some time of emergency, then it is extremelybeneficial to definitely be able to determine the safety of the productat the time that it is to be used.

One particularly important feature of the biological material detectingsystem of the instant invention is its ability to quantitativelysensitize the reagents so as to visually identify only those biologicalmaterials which have reached a predetermined concentration or thresholdlevel which is deemed to be harmful to humans.

For example, almost all poultry meat contain traces of the salmonellabacteria. In most cases, the salmonella levels have not reached aharmful level of concentration. The biological material detectingreagents are designed to visually report only those instances where thelevel of concentration of biological materials are deemed harmful byhealth regulatory bodies.

The method of production of the biological material detecting system isdesigned to be easily incorporated within the packaging infrastructureof existing systems without disruption of the systems or the proceduresunder which they are operating. The biological material detecting systemcan be incorporated onto packaging films which are produced by thepackager, or those which are supplied by a film manufacturer. Theapparatus necessary for applying the biological material detectingsystem may be easily located at the beginning of any continuous processsuch as printing or laminating and will operate as an integral part ofan existing system.

The biological material detecting system of the instant inventionrepresents an entirely new packaging material which is designed toinform the consumer of the presence of certain biological materials orpathogens present in food stuffs or other materials packaged within thedetecting system. The system is designed so that the presence of abiological material is presented to the consumer in a distinct,unmistakable manner which is easily visible to the naked eye.

Recent outbreaks of E. Coli and other health hazards have presentedserious problems to the general population and have raised concernsregarding the safety of the food supply.

It is an objective of the present invention to provide a biologicalmaterial detecting system for protecting the consumer by detecting andunmistakably presenting to the untrained eye visual icons on thepackaging material which signify the presence of a number of pathogensin the food stuff or other materials which are at a level harmful tohumans.

It is another objective of the instant invention to provide a bioassaymaterial wherein an antigen detecting antibody system is immobilizedupon the surface of a flexible polyolefin film.

It is still another objective of the instant invention to provide abioassay material wherein an antigen detecting antibody system isimmobilized upon the surface of a flexible polyvinylchloride film.

It is a further objective of the invention to provide a biologicalmaterial detecting system which is so similar in appearance andutilization that its use, in lieu of traditional packaging materials, isnot apparent to the food processor or other packagers.

A still further objective of the present invention is to provide abiological material detecting system which is cost effective whencompared to traditional packaging materials.

Other objectives and advantages of this invention will become apparentfrom the following description taken in conjunction with theaccompanying drawings wherein are set forth, by way of illustration andexample, certain embodiments of this invention. The drawings constitutea part of this specification and include exemplary embodiments of thepresent invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cross-sectional interpretation of an antibody sandwichimmunoassay device;

FIG. 2 is a cross-sectional interpretation of a single ligand assay;

FIG. 2A is a cross-sectional interpretation of a single ligand assayincluding a chromogenic ligand;

FIG. 3 is a diagrammatic representation showing the functioning of asingle ligand assay;

FIG. 4 is a cross-sectional interpretation of an antibody sandwichimmunoassay including a scavenger system for microbial quantification;

FIGS. 5 and 6 are a diagrammatic representation showing the functioningof a sandwich assay/scavenger system;

FIG. 7 is a planar view of an example of icon placement and printing;

FIG. 7A is an example of a typical code of identification applied to theicon pattern;

FIG. 8 is the result derived from EXAMPLE 2 and exemplifies capturesensitivity of a single ligand treated polyvinylchloride film;

FIG. 9 is a block diagram of the apparatus illustrating the processsteps for forming a sandwich assay;

FIG. 10 is a block diagram of the apparatus illustrating the processsteps for forming a single ligand assay.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

Referring now to FIG. 1, the detection and identification of variousbiological materials in packaged foods or other products is accomplishedby the use of antibodies which are specific to the biological materialbeing sought. Specific antibodies, defined as capture antibodies, arebiologically active ligands characterized by their ability to recognizean epitope of the particular toxic substance being tested for. Thesecapture antibodies are selected from such materials as antibodies,aptamers, single stranded nucleic acid probes, lipids, naturalreceptors, lectins, carbohydrates and proteins. In one embodiment of theinvention, the capture antibodies are arranged with unique icon shapesand in particular patterns. The capture antibodies are immobilized tothe polymer film. An agarose gel coat containing detector antibodies isprinted in register above the capture antibodies. A protective gel coatcompletes the construction of the packaging material. The gel coatconstituting the inner layer, e.g. that layer which is next to thepackaged product, is a special type of gel coat or an equivalent theretowhich has sufficient porosity to allow toxic molecules, known asantigens, to migrate through it to an antibody “sandwich” laminatedbetween the polymer film and the gel coat. The special gel coat hassufficient abrasion resistance to prevent exposure of the reagents tothe product. The special gel coat useful in the invention is a readilyavailable coating commonly utilized in the food industry to coat candiesand the like, e.g. coated chocolates to prevent them from melting onone's hands. Migration of antigens is driven by capillary action andnormally reaches a state of equilibrium within a 72 hour time period. Ina particularly preferred embodiment, when operating within a temperaturerange of 4-25 degrees Celsius, an initial positive reading can beobtained within 30 minutes, and the test continues to yield results forabout 72 hours. Upon migrating through the special gel coat the antigenenters an agarose gel film which has surfactant properties, containsfree detector antibodies, and also contains one or more ingredientsdesigned to enhance the growth of microbial materials, e.g. nutrientssuch as sorbitol, NOVOBIOCIN, CEFIXIME and TELLURITE which increase thegrowth rate and ease isolation of E. Coli 0157H. If the antigenencounters a species of antibody which is specific to an epitopethereof, it will then bind to it forming a detector/antibody complex.Once bound thereto, the bound antigen/antibody complex becomes too largeto migrate back through the special gel coat due to its inherent fineporous structure. This insures that pathogenic material can not migrateback into the product being tested. Continuing pressure towardequilibrium from capillarity will tend to move the antigen, with itsbound antibody, through a second gel coat layer and into an area of theflexible polyvinylchloride or polyolefin film containing correspondingspecies of immobilized capture antibodies. The layer of immobilizedantibodies is attached to the outer polymer film in predeterminedpatterns of simple icons, as best seen in FIGS. 7, 7A. When theparticular species of bound antigen encounters a particularcorresponding species of immobilized antibody specific to a separate anddistinct epitope thereof, further binding occurs. Upon the antigenbinding to the two antibodies, a distinct icon shape emerges on theouter film at the point of binding, thereby providing a visualindicator.

While it is theoretically possible to detect an unlimited number ofpathogens present in a packaged product, then to present thisinformation in a very clear and unmistakable manner to an untrainedconsumer, as a practical matter there are limits to the amount ofinformation which can be developed and presented in the biologicalmaterial detecting system. Some of the limiting factors are cost,available surface area for display of information, complexity, and otherconsiderations. Thus, for illustrative purposes only, the biologicalmaterial detecting system as exemplified herein utilizes four separatepairs of antibodies, as set forth in FIGS. 7 and 7A. This is in no waymeant to suggest a limit on the number of antibodies that can beutilized in a single biological material detecting system. Asdemonstrated in FIGS. 7 and 7A, the invention is exemplified withreference to detection of the following four microbes:

1. E-Coli;

2. Salmonella;

3. Listeria; and

4. Cyclospora.

To each of the four microbes, a particular icon shape is assigned.Although there are infinite numbers of icons which might be usedincluding letters, numbers, or even words, we have chosen simpleidentifiers for the purpose of demonstration. As an initial step in theconstruction of the biological material detecting system, the outerpolymer film or base layer undergoes a printing process in which apattern of the four icons, wherein each icon utilizes a specific speciesof immobilized capture antibody, is applied thereto. Correspondingspecies of free antibodies, known as detector antibodies, which arebiologically active ligands characterized by their ability to recognizea different epitope of the same particular toxic substance being testedfor, and suspended in an agarose gel solution containing a surfactantand a nutrient, are printed in registration with the immobilizedantibodies so as to be in overlying and juxtaposed relationship thereto,and are then dried. Lastly, a second gel coat having a degree ofporosity sufficient to prevent passage of the detector antibodies islaminated to the preparation.

Although the detection of biological materials through the use ofantibodies is well known, there are several new and novel aspects to theapplication of antibody science which are set forth in the developmentof the biological material detecting system of the present invention.

Among these are: 1) the use of multiple antibodies to detect multiplebiological materials in individual packages; 2) the use of a distinctiveicon or other shape to not only detect, but visually identify thebiological materials to the consumer, vendor, regulator, etc.;3)insuring that detection and identification of the biological materialsis accomplished in a timely manner in each particular application byjudiciously controlling the porosity of the gel coat, therebycontrolling the lapse rate of the reaction through the strength ofcapillary action; 4) inclusion of additives within the special gel coatto enhance the levels of microbes present; 5) incorporating thebiological material detecting system of the instant invention within theexisting packaging industry infrastructure; and 6) providing a bioassaymaterial and methods for its production and use which immobilizes theantibodies onto the surface of a flexible polyvinylchloride orpolyolefin, e.g. a polyethylene, a surface treated polyethylene, apolypropylene, a surface treated polypropylene or mixture thereof.

The embodiment discussed above is based upon a sandwich immunoassay asdepicted in FIG. 1, which measures specific microbes, wherein theparticular toxic substance is one or more members selected from thegroup consisting of a particular microorganism, biological materialscontaining the genetic characteristics of said particular microorganism,and mutations thereof. In a particular embodiment, the toxic substanceis selected from the group consisting of microorganisms, nucleic acids,proteins, integral components of microorganisms and combinationsthereof. It should also be understood that the invention will functionby direct measurement of microbes with certain types of antibodies,selected from the group consisting of an antibody, a single strandednucleic acid probe, an aptamer, a lipid, a natural receptor, a lectin, acarbohydrate and a protein. The biological materials may also bemeasured by non-immunological methods in particular using labeledmolecules, such as aptamers, which have a high affinity for thebiological materials.

The invention utilizes various types of detector antibodies, e.g. thoseconjugated with dyes to produce a visual cue, or alternatively,photoactive compounds capable of producing a visual cue in response to aparticular type of light exposure, for example a scanning system whichdetects luminescent properties which are visualized upon binding of theantigen and antibody. In this method of construction biologicalmaterials are measured directly with a biologically active ligand, e.g.an antibody, aptamer, nucleic acid probe or the like, which induces aconformational change to produce a visual cue.

It is also understood that specific polymers may be incorporated intothe invention and that when a biological material is bound to thesurface it induces a molecular change in the polymer resulting in adistinctly colored icon.

Referring to FIGS. 2 and 2A, in an alternative embodiment asandwich-type of construction is not necessary. As depicted in FIGS. 2and 2A, the provision of certain types of biologically active ligand,e.g. chromogenic ligands to which receptors are bound will permit thevisual confirmation of binding of the antigen to the immobilized ligand.

As depicted in FIG. 3, a polymer film is provided and a biologicallyactive ligand, preferably a chromogenic ligand, is immobilized to thepolymer film. In the past, immobilized ligands were attached to rigidsolid support matrices such as plastic, polystyrene beads, microtitreplates, latex beads, fibers, metal and glass surfaces and the like. Theimmobilized ligands have also been attached to flexible surfaces such asnitrocellulose or polyester sheets which were not transparent.Surprisingly, the inventor has discovered that it is possible to attachbiologically active ligands to the surface of various flexible polymericfilms, for example polyvinylchloride and polyolefins, e.g. a polyolefinsheet having appropriate properties of transparency and flexibility andthat the composite functions as a biological sensor or assay material.After printing on the polymer film, the material goes through a dryingstep; subsequent to which a special gel coat or liquid film is appliedas a protectant layer and the final product is then dried.

Illustrative of films which will function in the present invention is afilm containing a structural polymer base having a treated surface andincorporating therein a fluorescing antibody receptor and finally astabilized gel coat. These films may be untreated polyethylene orpolyvinylchloride films which are amenable to antibody immobilization byvarious mechanisms, e.g. by adsorption. In a particular embodiment, thefilms may be first cleaned, e.g. by ultrasonication in an appropriatesolvent, and subsequently dried. For example the polymer sheet may beexposed to a fifteen minute ultrasonic treatment in a solvent such asmethylene chloride, acetone, distilled water, or the like. In somecases, a series of solvent treatments are performed. Subsequently thefilm is placed in a desiccating device and dried. Alternatively, thesefilms may be created by first exposing the film to an electron dischargetreatment at the surface thereof, then printing with a fluorescingantibody receptor. Subsequently, a drying or heating step may beutilized to treat the film to immobilize the receptor. Next, the film iswashed to remove un-immobilized receptor; the film is then coated with agel and finally dried.

Additional modifications to polyolefin films may be conducted to createthe presence of functional groups, for example a polyethylene sheet maybe halogenated by a free radical substitution mechanism, e.g.bromination, chlorosulfonation, chlorophosphorylation or the like.Furthermore, a halodialkylammonium salt in a sulfuric acid solution maybe useful as a halogenating agent when enhanced surface selectivity isdesirable.

Grafting techniques are also contemplated wherein hydrogen abstractionby transient free radicals or free radical equivalents generated in thevapor or gas phase is conducted. Grafting by various alternative meanssuch as irradiation, various means of surface modification, polyolefinoxidation, acid etching, inclusion of chemical additive compounds to thepolymer formulation which have the ability to modify the surfacecharacteristics thereof, or equivalent techniques are all contemplatedby this invention.

Additionally, the formation of oxygenated surface groups such ashydroxyl, carbonyl and carboxyl groups via a flame treatment surfacemodification technique is contemplated.

Further, functionalization without chain scission by carbene insertionchemistry is also contemplated as a means of polyolefin polymermodification.

Illustrative of the types of commercially available films which might beutilized are polyvinylchloride films and a straight polyethylene filmwith electron discharge treatment marketed under the trademark SCLAIR®.The electron discharge treatment, when utilized, renders the film muchmore susceptible to immobilization of the antibodies on its surface.Additional films which might be utilized are Nylon 66 films, for exampleDARTEK®, a coextrudable adhesive film such as BYNEL® and a blend ofBYNEL® with polyethylene film.

With reference to FIGS. 4-6, one of the most important features of thebiological material detecting system is its ability to quantitativelysensitize the antibody or aptamer so as to visually identify only thosebiological materials that have reached a concentration level deemedharmful to humans. One means of providing this sensitization is byincluding a scavenger antibody which is a biologically active ligandcharacterized as having a higher affinity for the particular toxicsubstance than the capture antibody. The scavenger antibody is providedin a sufficient amount to bind with the particular toxic substance up toand including a specific threshold concentration. In this manner, thecapture antibody will be prevented from binding with a detector antibodyuntil the concentration of the particular biological material surpassesthe specific threshold concentration. In this manner, the biologicalmaterial detecting system visually reports only those instances whereconcentration levels are deemed harmful by health regulatory bodies.

Since the biological material detecting system as described herein canmaintain its activity over long periods of time, e.g. up to 1 year, itis able to protect against contamination in products which have longshelf lives. Additionally, by reporting only toxic concentrations, itavoids “false positives” and, in some cases, can extend the useful lifeof the product.

Referring to FIGS. 9 and 10, the apparatus for producing the biologicalmaterial detecting system is illustrated. These embodiments areessentially particular combinations of printers, coaters and dryerswhich will be used to place biologically active reagents upon a thinpolymer film useful for packaging food stuffs and other products. Theinstant invention further includes the fabrication of such a film in theform of sealable or resealable bags, e.g. bags having a foldable orzipper-like closure, or the like closure for effecting secure retentionof the contents. In certain embodiments the bag may be heat sealed toinsure against tampering or to maintain a sterile environment or thelike. These films will be further processed subsequent to application ofthe biological material detecting system by printing, laminating, orequivalent methods of fabrication. The machinery is designed so that itwill transport and process very thin films at rather high speeds.Furthermore, the machinery is designed so that it can be utilizedeffectively as an additional processing step when added to continuousprocessing operations already in use at packaging material fabricationplants. The printing machinery is designed so that a minimum of fourdistinct biological active ligands in a hydrate solution can be printedin patterns in a precise registration on the polymer film. The printingmay be accomplished by jet spray or roller application, or equivalentprinting methods. Each print applicator is capable of printing adetailed icon no larger than ¼″×¼″ in a minimum thickness. Patterningmay be controlled by computer or roller calendaring. It is important todetermine the appropriate viscosity of the solution to be applied sothat successful printing, coating, and drying can be accomplished. Afterthe printing step the icons must be protected. This is accomplished by afinal application of a thin special gel coat or a thin liquid film. Thisstep is accomplished by a 100% coating of the entire film oralternatively by selectively coating each icon such that a 10% overlapis coated beyond the icon in all directions. This coating step may beaccomplished with sprays or rollers and the viscosity of the coatingmaterial must be optimized so as to provide adequate coverage. Thebiological material detecting system must be dried after printing andonce again after coating. The drying is accomplished in a very rapidmanner so as to enable high through put for the process. Various meansof drying include the use of radiant heat, convected air and freezedrying. Care must be taken to avoid drying temperatures which willinactivate the biological reagents which have been applied. The polymerfilm which has been surface treated in the form of electron discharge,e.g. corona treatment, is most preferred. After preparation, the thinfilm is transported at relatively high speeds so that a wrinkle freesurface is provided for printing, coating and rollup. Additionally, theapparatus provides a complete recovery system for the reagents whichallows for total recovery of the agents and the volatile organiccontaminants.

The invention will be further illustrated by way of the followingexamples:

EXAMPLE 1

Detection of Antibody on the Surface of a Thin Layer PolyvinylchlorideSheet:

Rabbit polyclonal IgG was diluted to a final concentration of 2.0 μg/mlin 0.1 M carbonate (Na₂CO₃)-bicarbonate (NaHCO₃) buffer, pH 9.6.

Using a 211″×3″ grid, 75 μL (150 ng) was applied to a sheet ofpolyvinylchloride at 1″ intervals.

The antibody treated polyvinylchloride sheet was dried for 1.5 hrs. at atemperature of 37° C.

The dried sheet was then washed 3 times with a phosphate buffered salinesolution at a ph of 7.4.

HRP conjugated goat anti-rabbit IgG (GαR^(HRP)) was diluted to aconcentration of 1:7000 in 1% casein, 0.1M potassium ferricyanideK₃Fe(CN)₆, 0.1% phosphate glass (Na₁₅P₁₃O₄₀-Na₂₀P₁₈O₅₅), at a pH of 7.4.

A precision pipette was used to apply 125 μL of diluted G^(HRP) to thegrid backed polyvinylchloride sheet at 1″ intervals coinciding with thearea covered by the previously coupled RαG.

The sheet was incubated at room temperature for 30 minutes.

The sheet was then washed 3× with phosphate buffered saline at a pH of7.4.

125 μL of precipitating TMB enzyme substrate was added to the testareas.

The sheet was incubated at room temperature until color development wascomplete.

Lastly the sheet was washed 3 times with deionized water and allowed toair dry.

EXAMPLE 2

Full Sandwich Immunoassay on the Surface of a Thin LayerPolyvinylchloride Sheet

Rabbit polyclonal IgG was diluted to a final concentration of 2.0 μg/mlin 0.1 M carbonate (Na₂CO₃)-bicarbonate (NaHCO₃) buffer, pH 9.6.

A 13×9 cm piece of thin layered polyvinylchloride sheet was insertedinto a BIO-RAD DOT-SPOT apparatus possessing 96 sample wells spaced at1.0 cm intervals in a 12×8 well grid.

A 100 μL sample (1.0 μg) of rabbit polyclonal IgG was applied to eachwell 8 of column 1.

Antibody samples applied to columns 2-12 represented serial dilutions ofthe antibody ranging from 500 ng-0.5 ng.

The antibody treated polyvinylchloride sheet was dried overnight at 37°C.

The dried sheet was washed 3 times with phosphate buffered saline (PBS),pH 7.4.

Antigen was diluted to a final concentration of 1.0 μg/ml in trisbuffered saline (TBS) with 1% casein, pH 7.4.

100 μL, representing 100 ng, of antigen, was applied to each well of theapparatus and incubated at room temperature for 1 hour.

The polyvinylchloride sheet was washed 3 times with phosphate bufferedsaline (PBS), pH 7.4.

Detector mouse monoclonal antibody was diluted was diluted 1:625 withTBS containing 1% casein, 0.1 M potassium ferricyanide K₃Fe(Cn)₆, and0.1% phosphate glass (Na₁₅P₁₃O₄₀-Na₂₀P₁₈O₅₅), pH 7.4.

100 μL of the 1:625 dilution of detector antibody solution was appliedto each well of row #1.

Detector samples of 100 μL applied to rows 2-7 represented serialdilutions of the antibody ranging from 1:1,250 to 1:80,000. Dilutions ofdetector antibody were incubated on the polyvinylchloride sheet for 1Hr. at room temperature.

The polyvinylchloride sheet was washed 3 times with phosphate bufferedsaline (PBS), pH 7.4.

100 μL of goat anti-mouse IgG^(HRP) were added to each well of theDOT-SPOT apparatus and allowed to incubate for one hour at roomtemperature.

The polyvinylchloride sheet was washed 3 times with phosphate bufferedsaline (PBS), pH 7.4.

100 μL of precipitating TMB enzyme substrate was added to the testareas.

The sheet was incubated at room temperature until color development wascomplete (see FIG. 8).

Lastly the sheet was washed 3 times with deionized water and allowed toair dry.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementof parts herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown in the drawings and described in thespecification.

What is claimed is:
 1. A biological assay material for detecting thepresence of a toxic substance comprising: a flexible base forimmobilization of a ligand applied to a surface thereof, said baseselected from the group consisting of polyolefin and polyvinylchloride;a permeable layer adjacent said flexible base and having a captureantibody thereon, said antibody being a biologically active ligandcharacterized by its ability to recognize an epitope of a toxicsubstance; and a protective layer adjacent said permeable layer andincluding a biologically active detector antibody which acts as a meansto selectively control the migration of antibodies and antigens, saiddetector antibody characterized by its ability to recognize an epitopeof a toxic substance forming an antibody/antigen complex; wherebypassage of a toxic substance is permitted and passage of saidantibody/antigen complex is prevented.
 2. The biological assay materialaccording to claim 1 wherein the flexible base is a polyolefin selectedfrom the group consisting of polyethylene, polypropylene and mixturesthereof.
 3. The biological assay material according to claim 1 whereinthe flexible base is a polyvinylchloride.
 4. The biological assaymaterial according to claim 1 wherein the toxic substance is one or moremembers selected from the group consisting of a microorganism,biological materials containing the genetic characteristics of saidmicroorganism, and mutations thereof.
 5. The biological assay ofmaterial according to claim 1 wherein the toxic substance is selectedfrom the group consisting of microorganisms, nucleic acids, proteins,integral components of microorganisms and combinations thereof.
 6. Thebiological assay material according to claim 1 wherein the ligand isselected from the group consisting of an antibody, a single strandednucleic acid probe, an aptamer, a lipid, a natural receptor, a lectin, acarbohydrate and a protein.
 7. The biological assay material accordingto claim 1 said permeable layer further including a scavenger antibodywhich is a biologically active ligand characterized as having a higheraffinity for the toxic substance than the capture antibody, saidscavenger antibody being present in a sufficient amount to bind with thetoxic substance up to and including a specific threshold concentration;whereby a capture antibody will be prevented from binding with adetector antibody until the concentration of the biological materialsurpasses the specific threshold concentration.
 8. A method to detectthe presence or absence of a toxic substance, which method comprises: a)providing a flexible base for immobilization of a ligand applied to asurface thereof, said base selected from the group consisting ofpolyolefin and polyvinylchloride; b) providing a permeable layeradjacent said flexible base and having a capture antibody thereon, saidantibody being a biologically active ligand characterized by its abilityto recognize an epitope of a toxic substance; c) further providing aprotective layer adjacent said permeable layer and including abiologically active detector antibody which acts as a means toselectively control the migration of antibodies and antigens, saiddetector antibody characterized bv its ability to recognize an epitopeof a toxic substance and thereby forming an antibody/antigen complex; d)placing said biological assay material in an environment which maycontain a toxic substance, whereby simultaneous binding of said captureantibody and said detector antibody with the particular toxic substancewhich they recognize results in the appearance of a visual signal; ande) monitoring said biological assay material for a period of timesufficient to observe said visual signal which will confirm the presenceor absence of a toxic substance.
 9. A material useful for food packagingand characterized by its ability to detect the presence and particularlyidentify one or more toxic substances comprising: a flexible base forimmobilization of a ligand applied to a surface thereof, said baseselected from the group consisting of polyolefin and polyvinylchloride;a permeable layer adjacent said flexible base and having a captureantibody thereon, said antibody being a biologically active ligandcharacterized by its ability to recognize an epitope of a toxicsubstance; and a protective layer adjacent said permeable layer andincluding a biologically active detector antibody which acts as a meansto selectively control the migration of antibodies and antigens, saiddetector antibody characterized by its ability to recognize an epitopeof a toxic substance forming an antigen/antibody complex; wherebypassage of a toxic substance is permitted and passage of saidantibody/antigen complex is prevented, said protective layer having adegree of abrasion resistance effective to protect the material.
 10. Thematerial according to claim 9 wherein the flexible base is selected fromthe group consisting of polyethylene, polypropylene and mixturesthereof.
 11. The material according to claim 9 wherein the flexible baseis a polyvinylchloride.
 12. The material according to claim 9 whereinthe toxic substance is one or more members selected from the groupconsisting of a particular microorganism, biological materialscontaining the genetic characteristics of said particular microorganism,and mutations thereof.
 13. The material according to claim 9 wherein thetoxic substance is selected from the group consisting of microorganisms,nucleic acids, proteins, integral components of microorganisms andcombinations thereof.
 14. The material according to claim 9 wherein theligand is selected from the group consisting of an antibody, a singlestranded nucleic acid probe, an aptamer, a lipid, a natural receptor, alectin, a carbohydrate and a protein.
 15. The material according toclaim 9, said permeable layer further including a scavenger antibodywhich is a biologically active ligand characterized as having a higheraffinity for the toxic substance than the capture antibody, saidscavenger antibody being present in a sufficient amount to bind with thetoxic substance up to and including a specific threshold concentration;whereby a capture antibody will be prevented from binding with adetector antibody until the concentration of the particular biologicalmaterial surpasses the specific threshold concentration.
 16. Thematerial according to claim 9 wherein one or more species of captureantibody are immobilized onto said surface of said flexible base in aparticular orientation, each of said one or more species beingcharacterized by a unique shape; and one or more corresponding speciesof detector antibody are applied onto the surface of said layer; wherebysimultaneous binding of any of the one or more species of captureantibodies and one or more corresponding species of detector antibodieswith the toxic substance which they recognize results in the appearanceof a visual signal having the unique shape assigned to that species;wherein an observer is alerted to the presence and identity of saidtoxic substance.
 17. A biological assay material for detecting thepresence of a particular toxic substance comprising: a flexible base forimmobilization of a ligand applied to a surface thereof, said baseselected from the group consisting of polyoleftn and polyvinylchloride;a biologically active ligand immobilized to the flexible base; and a gelcoat or liquid film applied as a protectant layer; wherein saidbiological assay material is a food packaging material in the form of aresealable bag; whereby simultaneous binding of said biologically activeligand and said particular toxic substance results in the appearance ofa visual signal; wherein said visual signal is indicative of both thepresence and identity of said toxic substance.
 18. The biological assaymaterial according to claim 17 wherein the biologically active ligand isa chromogenic ligand.
 19. The biological assay material according toclaim 17 wherein the flexible base is a film incorporating thereon afluorescing antibody receptor.
 20. The biological assay materialaccording to claim 19 wherein the flexible base is created by printingwith a fluorescing antibody receptor and drying or heating the film toimmobilize said receptor.
 21. The biological assay material according toclaim 17 wherein a scavenger antibody which is a biologically activeligand characterized as having a higher affinity for the toxic substancethan the immobilized ligand is further immobilized to said flexible baseand is provided in a sufficient amount to bind with the toxic substanceup to and including a specific threshold concentration; whereby theassay material is quantitatively sensitized so as to visually identifyonly those toxic substances that have reached a concentration leveldeemed harmful to humans.
 22. The biological assay material according toclaim 18 wherein the chromogenic ligand is selected from the groupconsisting of those conjugated with dyes to produce a visual cue andthose characterized as photoactive compounds capable of producing avisual cue in response to a particular type of light exposure; wherebybinding of the toxic substance and chromogenic ligand results in a colorchange or visualization of a luminescent property which is indicative ofboth the presence and identity of said toxic substance.
 23. Thebiological assay material according to claim 17 containing a pluralityof biologically active ligands, each of said ligands being receptive toan epitope of a different toxic substance and having a unique shape;whereby upon binding with one or more of said different toxicsubstances, a visual signal will result thereby alerting an observer tothe presence and identity of any or all of the toxic substances to whichsaid material is receptive.
 24. The biological assay material accordingto claim 17 wherein the toxic substance is one or more members selectedfrom the group consisting of a particular microorganism, biologicalmaterials containing the genetic characteristics of said particularmicroorganism, and mutations thereof.
 25. The biological assay ofmaterial according to claim 17 wherein the toxic substance is selectedfrom the group consisting of microorganisms, nucleic acids, proteins,integral components of microorganisms and combinations thereof.
 26. Thebiological assay material according to claim 17 wherein the ligand isselected from the group consisting of an antibody, a single strandednucleic acid probe, an aptamer, a lipid, a natural receptor, a lectin, acarbohydrate and a protein.
 27. The material according to claim 17wherein the flexible base is selected from the group consisting ofpolyethylene, polypropylene and mixtures thereof.
 28. The materialaccording to claim 17 wherein the flexible base is a polyvinylchloride.29. The material according to claim 1 wherein the biologically activeligand is of plant origin.